The long-term goal of the proposed research is a better understanding of the role of copper metallothionein (CuMT) in copper metabolism and in metalloenzyme activation. It is hoped that more detailed knowledge of the components involved in copper-metalloprotein biosynthesis may provide insight into the molecular bases of metabolic disorders that arise from errors in copper metabolism and from decreased activities of copper- dependent enzymes, such as Wilson's disease and Menkes' disease. The more immediate goal of the proposed research is to explore the role of CuMT in the molecular events that lead to the insertion of copper into apohemocyanin (apoHc). In vitro studies on the activation of apoHc will be carried out with a specific metallothionein, CuMT-3, that we have recently purified from the hepatopancreas of the American lobster and the blue crab. In the latter organism there is a direct correlation between levels of Cu(I)MT and Cu(I)- Hc during its molt cycle. This provides us with a unique model system for the study of the involvement of MTs in metalloprotein activation and degradation on a cellular and organismal level. We will establish whether the in vitro activation of apoHc by CuMT-3 requires a direct interaction between the two proteins or whether free Cu(I) in solution is involved in the copper transfer process. We will study the molecular events involved in Hc activation, and the effect of the molting hormone, 20 hydroxyecdysone, on these processes, using suspensions of hepatopancreas cells and radiolabeled forms of copper and cysteine. CuMTs can be separated into multiple forms, and preliminary evidence suggests that these different forms may have different biological functions. As a step towards further clarifying this possibility we will determine if the form of MT which sequesters copper as Hc is broken down is different from the MT that acts as a copper donor during Hc synthesis. Our proposed studies will specifically increase our knowledge of the biological function(s) of the different forms of CuMT and are of general importance to a better understanding of the mechanism of copper insertion into apometalloproteins.